Project Summary Selective estrogen receptor downregulators (SERDs) are a class of endocrine therapy agents that act both as estrogen receptor (ER) antagonists and ER degraders effective in treating metastatic or advanced breast cancer that disproportionately affects African American women. Fulvestrant is the only FDA approved SERD indicated for advanced or metastatic breast cancer both as a first line and second line endocrine agent. However, this injection only drug is poorly bioavailable and it takes 30 days to reach its maximal steady-state plasma concentration, limiting the clinical response rate to lower than 20% in the hormone resistant setting. An oral SERD with greater drug exposure and faster action would bring immediate clinical benefits to patients with advanced breast cancer. Further, in light of the recent FDA approval of fulvestrant as a combination therapy with CDK4/6 inhibitor palbociclib for advanced breast cancer, the clinical utility of an oral SERD in the combination treatment setting is also very significant. Advances in oral SERDs development have been limited to nonsteroidal molecules with several being currently evaluated in phase 1 clinical trials, yet none has advanced to phase II clinical trials. Our lead compound, ZB716, has shown promising preclinical data in bioavailability, efficacy, and toxicology. ZB716 binds to ER with high affinity and exerts its antiestrogenic effect on ER-expressing breast cancer cells. In both tamoxifen naive and tamoxifen resistant breast cancer cells, ZB716 potently inhibits cell proliferation and effectively degrades the hormone receptor in a dose-dependent manner. In animals, we have shown that ZB716 has far superior oral bioavailability when compared to fulvestrant. Moreover, in direct comparison to the two oral SERDs under clinical trials, ZB716 is a stronger antiestrogen and ER-degrader. To further advance the preclinical development of ZB716 we propose to investigate the in vivo efficacy of ZB716 in endocrine resistant, patient derived breast tumor models that most closely resemble clinical settings for which SERD is indicated. We will also evaluate ZB716 efficacy in combination with a CDK4/6 inhibitor, palbociclib and investigate the mechanism of action of ZB716 on patient-derived xenografts (PDX) expressing mutant forms of ER and determine the binding behavior of ZB716 to mutant ERs and its modulation of ER?-coregulator interactions. Finally, we will determine optimal reaction conditions under which ZB716 can be prepared in larger scale, investigate its physical properties and formulation options for toxicological studies in animals, and conduct metabolic profiling, pharmacokinetics, and bioavailability studies. Accomplishing the proposed studies will provide key efficacy data to determine whether ZB716 is effective in treating endocrine resistant, ESR1 mutant breast cancer and whether it is a true antiestrogen and ER degrader by acting through the ER. The studies will also demonstrate the clinical utility of ZB716 as a combination therapy when used with a CDK4/6 inhibitor. Moreover, synthetic method optimization will pave the way for scalable manufacture of the API, and safety pharmacology and physical chemical properties will fulfill IND-enabling data. In summary, the proposed research will advance this promising oral SERD towards clinical trials to test its safety and efficacy in breast cancer patients.